Opposing Changes in 3α-Hydroxysteroid Dehydrogenase Oxidative and Reductive Activities in Rat Leydig Cells during Pubertal Development1

Abstract

The enzyme 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) has an important role in androgen metabolism, catalyzing the interconversion of dihydrotestosterone (DHT) and 5alpha-androstane-3alpha,17beta-diol (3alpha-DIOL). The net direction of this interconversion will affect the amount of biologically active ligand available for androgen receptor binding. We hypothesize that in Leydig cells, differential expression of 3alpha-HSD enzymes favoring one of the two directions is a mechanism by which DHT levels are controlled. In order to characterize 3alpha-HSD in rat Leydig cells, the following properties were analyzed: rates of oxidation (3alpha-DIOL to DHT) and reduction (DHT to 3alpha-DIOL) and preference for the cofactors NADP(H) and NAD(H) (i.e., the oxidized and reduced forms of both pyridine nucleotides) in Leydig cells isolated on Days 21, 35, and 90 postpartum. Levels of 3alpha-HSD protein were measured by immunoblotting using an antibody directed against the liver type of the enzyme. Levels of 3alpha-HSD protein and rates of reduction were highest on Day 21 and lowest on Day 90. The opposite was true for the rate of 3alpha-HSD oxidation, which was barely detectable on Day 21 and highest on Day 90 (59.08 +/- 6.35 pmol/min per 10(6) cells, mean +/- SE). Therefore, the level of 3alpha-HSD protein detectable by liver enzyme was consistent with reduction but not with oxidation. There was a clear partitioning of NADP(H)-dependent activity into the cytosolic fraction of Leydig cells, whereas on Days 35 and 90, Leydig cells also contained a microsomal NAD(H)-activated 3alpha-HSD. We conclude that 1) the cytosolic 3alpha-HSD in Leydig cells on Day 21 behaves as a unidirectional NADPH-dependent reductase; 2) by Day 35, a microsomal NAD(H)-dependent enzyme activity is present and may account for predominance of 3alpha-HSD oxidation over reduction and the resultant high capacity of Leydig cells on Day 90 to synthesize DHT from 3alpha-DIOL.

Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

“…Microsomal preparations of human and rat testes were performed as described previously [14]. Briefly, testes were homogenized in cold 0.01 mmol L −1 phosphate buffered saline (PBS) containing 0.25 mmol L −1 sucrose and centrifuged at 700 × g for 30 min.…”

Influence of mirtazapine on plasma concentrations of neuroactive steroids in major depression and on 3α-hydroxysteroid dehydrogenase activity

“…18 3a-HSD activity has been described in the cytosolic and microsomal fractions of a number of human and animal tissues. 17,[19][20][21][22][23][24][25] To date, at least four types of cytosolic 3a-HSDs have been identified in humans (also called AKR1C1-AKR1C4), which display 3-, 17-, and 20-ketosteroid reductase activity. All of these cytosolic enzymes exhibit a preference for the phosphorylated nucleotides as cofactors (NADP þ , NADPH).…”

Characterization of a Novel Type of Human Microsomal 3α-Hydroxysteroid Dehydrogenase

“…In the central nervous system, 3␣-hydroxysteroid oxidoreductase activity controls the amount of a potent neurosteroid, 3␣-tetrahydroprogesterone (also called allopregnanolone), which serves as an allosteric regulator of all ␥-aminobutyric acid type A receptors and potentiates ␥-aminobutyric acid mediated chloride conductance (1). 3␣-Hydroxysteroid oxidoreductase activity has been described in the cytosolic and microsomal fractions of a number of human and animal tissues (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). In addition to the different subcellular localization, cytosolic and microsomal enzymes appear to have a distinctively different cofactor preference.…”

“…In addition to the different subcellular localization, cytosolic and microsomal enzymes appear to have a distinctively different cofactor preference. Cytosolic 3␣-hydroxysteroid oxidoreductases exhibit a preference for the phosphorylated nucleotides as cofactors (NADP ϩ /NADPH), whereas microsomal enzymes prefer NAD ϩ /NADH (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). Because the predominant forms of nucleotide cofactors in the cells are NAD ϩ and NADPH, it is generally believed that the NAD ϩ -dependent dehydrogenases function mainly in the oxidative direction, whereas the NADPH-dependent enzymes function as reductases (12).…”